Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system and is the commonest chronic neurological disease of young adults. The incidence of MS and its pattern of distribution have been unchanged for decades. The disease remains essentially untreatable.
MS usually affects multiple areas of white matter in the central nervous system (CNS), most frequently, the preventricual white matter, brain stem, spinal cord and the optic nerves. The primary process destroys myelin sheaths and eventually kills oligodendrocytes creating the characteristic plaque of MS.
The early development of the plaque is characterised by the development of perivascular inflammation followed by the migration of lymphocytes, plasma cells and macrophages into the lesion. This is followed by astrocyte gliosis and the attempts of demyelination by oligodendrocytes. The plaque is surrounded by lymphocytes.
Although the aetiology of MS is still unknown, the focus of research efforts that have led to plausible hypotheses have been those of immune dysregulation including auto-immunity and genetic predisposition, both of which may play a role in he actual development of disease. Both TNF.beta. (lymphotoxin) and TNF.alpha. are thought to play a role in the pathophysiology.
Multiple immunological abnormalities are reproducibly found in patients in the acute stage of the disease. The synthesis of immunoglobulins, although normal in the periphery, is increased in the central nervous system and the antibodies produced have a characteristic banding pattern. The antigenic specificity of these antibodies is not known and it is unclear whether they have a role to play in the progression of the disease.
Various stressors known to activate the immune system such as viral infection or surgery can also produce an exacerbation of MS. Other activator such as .gamma.-interferon produce similar effects when administered. In addition, immunosuppressive anti-inflammatory therapy with corticosteroids for example, can produce modest remission or at lease palliation for short periods of time, although this therapy is controversial.
Lymphocyte reactivity against two neuronal antigens, myelin basic protein and proteolipid has been demonstrated. Although not proven, this activity would form the basis for an auto-immune response against neuronal tissue.
Myelopathy, a disorder of the spinal cord, can have many different aetiologies, most of which are mediated by inflammation, including the following:
Neurosyphillis; PA1 b.sub.12 or folate deficiency; PA1 sarcoidosis; PA1 transverse myelitis; PA1 arachidonitis; PA1 cervical spondylitis; PA1 motor neuron disease; PA1 neurofibromatosis; PA1 spinal cord compression from tumour, disc or arthritis; PA1 lupus erythematosus of the spinal cord; and PA1 viral encephalomyelitis
Chronic inflammation or, as more commonly known, chronic immune system activation occurs in response to persistent antigen whose origin may be exogenous or may result from an auto-immune state. Such chronic inflammation results in local tissue destruction and depending upon the type of inflammation can result in systemic effects due to the sustained production of inflammatory mediators. Such inflammatory mediators include the cytokines which are soluble mediators produced by activated lymphocytes and macrophages and effect cellular communication and physiological response. Chronic immune activation can occur as a result of infectious disease, such as chronic fatigue syndrome or toxic shock syndrome or through auto-immune mechanisms resulting in such conditions as rheumatoid arthritis, inflammatory bowel disease, Crohns Disease and other diseases such as graft versus host disease.
Rheumatoid arthritis (Marrow et al, I "Auto-immune Rheumatic Disease", Blackwell Scientific Publ. Oxford, UK, Chapter 4 pp148-207 (1987) is a disease characterised by chronic inflammation and erosion of joints that may affect up to 3% of the population, including children. Symptoms of rheumatoid arthritis include morning stiffness, swelling and pain upon motion in at lease one joint and joint swelling. Non-specific symptoms including lethargy, anorexia and weakness as well as fever and lymphadenopathy (characteristic of immune activation) may antedate joint involvement. Extr.alpha.-articular manifestations of rheumatoid arthritis include vasculitis, cataracts, uveitis, interstitial fibrosis, pericarditis and myocarditis, peripheral neuropathy, myeloid deposits, chronic anaemia and subcutaneous and pulmonary nodules.
Genetic factors and infectious agents including bacteria, fungi, mycoplasmas and viruses have been associated with the development of rheumatoid arthritis. Mild rheumatoid arthritis may be treated with non-steroidal anti-inflammatory drugs while severe cases require systemic corticosteroids, anti-metabolites or cytotoxic agents. Experimentally, anti-CD4 monoclonal antibodies and anti-TNF.alpha. antibodies have been used to treat rheumatoid arthritis (Horneff et al, Cytokine 3 266-267 (1991); Horneff et al, Arth. Rheum. 34 129-140 (1991) and Shoenfeld et al, Clin. Exp. Rheum. 9. 663-673 (1991), Williams et al, 1992 PNAS 89, 9784).
Arthritis is the most common manifestation of systemic lupus erythematosus (SLE) and is classically the symptom for which the patient seeks medical attention. SLE is a set of clinical disorders involving multiple organ systems and characterised by the production of auto-antibodies and immune complexes. The immune complexes are deposited in various organs eg kidney, gastrointestinal tract and the resultant inflammation and tissue injury cause cell and organ dysfunction. B cell hyper-reactivity and production of auto-antibodies is an attributed imbalance between CD8 and CD4 T cells and altered cytokine production including increased production of interleukini 1, interleukin 2 and interferon .gamma.. Azathioprine is commonly used in management of the disease.
Systemic sclerosis (scleroderma) is characterised by thickening and fibrosis of the skin and by distinctive forms of internal organ involvement. (Claman, H. N., 1989, JAMA 262: 1206). The skin thickening of scieroderma is due to the accumulation of collagen in the lower dermis. The immobility of the skin is caused by replacement of the subcutaneous tissue with fibrous bands. Dermal fibroblasts obtained from involved skin accumulated types I, III and IV collagen, fibronectin and glycosaminoglycan. This is a secondary response to factors released by other cells. T-helper cells are demonstrable in involved skin. Elevated levels of serum IL-2 and IL-2 receptor are present and correlate with clinical progression of the disease (Kahaleh et al, 1989. Ann. Itern. Med. 110:446). This suggests a role for lymphokines that either stimulate collagen biosynthesis in fibroblasts or stimulate other cells such as monocytes or mast cells to produce such factors. Human chronic graft vs host disease is associated with a similar dermal fibrotic change. Interferon .gamma. is known to stimulate collagen accumulation by systemic scleroderma fibroblasts.
Polymyositis is a chronic inflamniatory disease of skeletal muscle, characterised by symmetric weakness of proximal limb girdle muscles and muscles of the trunk, neck and pharynx. A characteristic rash may also be present (Hochberg et al, 1986, Semin Arthritis Rheum 15:168). Peripheral blood lymphocytes from patients with polyin-yositis produce lymphokines cytotoxic to foetal muscle cells in vitro. In addition, the proportion of activated lymphocytes is increased particularly in muscle tissue. Environmental factors, particularly infectious agents (influenza B1, cocksackie virus B. Toxoplasma gondii), may be involved in the pathogenic process, Many drugs have been implicated in chronic inflammatory myopathy, including D-penicillamine, colchanicine, and ethanol.
Leukoclastic vasculitis (Laken and Smiley, 1981, Dis Mon. 64:181) is the name given to the pathologic lesions seen in the blood vessels that produce palpable purpura. It is thought that the lesions are produced by the interaction of cells and humoral products. The putative antigen (arising from streptococcal infection or drug interaction ) reacts with IgE attached to the surface of mast cells. The activated mast cells release factors (PAF, prostaglandins, leukotrienes) causing activation of platelets and release of vasoactive amines responsible for dilation and oedema in post capillary venules. Neutrophils migrate to the site, releasing toxic granule contents resulting in fibrinoid necrosis, extravasation of the red blood cells, and then to clinically apparent lesions of palpable purpura. In a related condition, Panniculitis, there is also inflammation of fatty tissue. There are two types of Panniculitis, septal and lobar. Among the septal forms, Behscets Disease may be a cause of erythema nodosum.
Inflammatory bowel disease (IBD) and Crohns disease are chronic inflammatory conditions that fulfil some of the criteria of an auto-immune disease (Snook, Gut 31 961-963 (1991)). Inflammation and tissue damage involves the recruitment and activation of neutrophils, macrophages and lymphocytes (MacDermott et al, Adv. Immunol. 42 285-328 (1988)) which generate cytokines and proinflammatory molecules such as prostaglandins and leukotrienes (MacDermott, Mt. Sinai J. Med. 57 273-278 (1990)). As a result of chronic activation of immunocompetent cells, IL-1, IL-6 (Starter, Immunol. Res. 10 273-278 (1990); Fiocchi, Immunol. Res. 10 239-246 (1991)) and TNF.alpha. (MacDermott, Mt. Sinai J. Med. 57 273-278 (1990)) are all elevated in IBD and Crohins Disease patients.
Drugs used to treat IBD and Crohns Disease include anti-inflammatory agents such as sulphasalazine (5-ASA) corticosteroids, cyclosporin A and azathiprine (Hanauer, Scand, J, Gastroenterol. 25 (Supl. 175) 97-106 (1990); Peppercorn, Annal. Intern. Med. 112 50-60 (1990)). Experimentally, anti-CD4 and anti-TNF monoclonal antibodies have been used to successfully treat ulcerative colitis (Emmerich et al, Lancet 338 570-571 (1991)).
Transplanted organs and tissues may be rejected when host T cells come in contact with donor HLA molecules. The process of rejection involves several effector mechanisms including cytotoxic T cells, lymphokines (principally TNFcc, IL-1, IL-2 and interferon y) and other soluble mediators of inflammation (eg prostaglandins and leukotrienes). Immunosuppressive drugs (eg azathrioprine, cortocisteroids, cyclosporine A, FK506, and polyclonal and monoclonal antibodies) inhibit T cell proliferation and production of cytokines in attempting to prolong graft survival.
Whilst a host may react against a genetically incompatible graft producing a host-versus-graft response, an immunocompetent graft (such as bone marrow or intestinal tissue) may react against the host resulting in graft-versus-host disease. These reactions are mediated by allogenic responses directed against a foreign MEC molecule and are mimicked in vitro by the mixed lymphocyte reaction (MLR). Graft/host interactions result in chronic inflammation surrounding the grafted tissue with an increase in markers of immune activation such as are seen in AIDS (Grant, Immunol. Today 12 171-172 (1991)). Treatment of the graft/host interactions currently include either azathioprine, cyclosporin A or methylprednisone and, more recently, rapamycin (Spekowski et al, Transplantation 53 258-264 (1992); Huber et al, Bibliotheca Cardiologica. 43 103-110 (19883). Monoclonal antibodies specific for CD3 (Wissing et al, Clin Exp Immunol. 83 333-337 (1991)), CD4 (Reinke et cd, Lancet 338 702-703 (1991)) and TNF.alpha. have been used experimentally to inhibit graft/host reactions.
Immediate hypersensitivity occurs after the binding of antigen to preformed antibodies of the IgE isotype bound to Fc receptors in mast cells or basophils. This binding leads to rapid degranulation and release of inflammatory mediators that act on tissues. The production of IgE is stimulated by T lymphocytes and their products (cytokines), mainly by interleukin 4 in synergy with interleukin 5, together with interleukin 6 and TMF produced by macrophages. Asthma, triggered by inhalant allergens, is an IgE mediated disease. Allergic rhinitis is an IgE-mediated inflammatory disease involving the nasal membranes. As many as 80% of patients with allergic asthma have coexistent symptoms of allergic rhinitis, and at least 40% of patients with allergic rhinitis manifest asthma at some time. In these conditions mast cells release histamine, leukotrienes such as LTB.sub.4, C.sub.4, D.sub.4 and E.sub.4, prostaglanidin D.sub.2 and proteases which are thought to be responsible for the immunopathology including an inflammatory infiltrate comprised largely of eosinophils. The presence of large numbers of neutrophils are also characteristic of asthmatic tissue.
Although the pathogenesis of atopic dermatitis is poorly understood, the frequent co-incidence of atopic dermatitis and allergic rhinitis or asthma suggests an immediate hypersensitivity may be involved. Serum IgE levels are frequently elevated in patients with atopic dermatitis and often decrease during periods of remission. Topical corticosteroid preparations are used during acute episodes and may be needed chronically in some patients. As a consequence, during exacerbations of eczema, patients frequently develop secondary bacterial infections.
Allergic contact dermatitis is a clinically important example of a delayed type hypersensitivity reaction which is T cell mediated involving release of T cell derived cytokines and proliferation of T cells within the skin. Other inflammatory cells, macrophages, lymphocytes, basophils, mast cells and eosinophils are also recruited to the involved area by cytokines and chemoattractants. Frequent contact sensitisers include Rhus antigen (found in poison ivy and oak), parapheylenediamine, nickel, rubber compounds, ethylenediamine, certain local anaesthetics (eg benzocaine), chromate and neomycin. Steroids, including prednisone, are employed for widespread contact dermatitis.
Psoriasis (Anderson and Voorhees, Psoriasis, In: Thiers, Dobson eds. Pathogenesis of skin disease. New York. Churchill Livingstone, 1986: 67) is a primary disease of the skin characterised bv well demarcated, inflammatory papules and plaques, which are typically covered by thickened scales. Neutrophils are found in psoriatic lesions. It is likely that arachidonic acid, levels of which are much higher than normal in psoriatic plaques, and its metabolites are important in this aspect of the disease. Further drugs which block the cyclo-oxygenase pathway of arachidonic acid metabolism (eg non-steroidal anti-inflammatory drugs such as indomethacin, phenylbutazone, meclotenamate) induce exacerbations of psoriasis. Dramatic clearing of recalcitrant, severe psoriasis has been achieved using cyclosporin A. The major mechanism of action of cylcosporin A is to inhibit the release of lymphokines produced by activated T lymphocytes. Thus it has been hypothesised that the activated T cells, in response to autologous or exogenous antigens, release factors that directly result in inflammation and epidermal proliferation, or indirectly produce these effects by activating macrophages or keratinocytes which then release cytokines, mediators of inflammation or growth factors that can elicit the pathology of psoriatic plaques.
Inflammation may be caused by bacteria, viruses and /or other infective agents, opportunistic infections (which may be consequent on an immunodepressed state, for example resulting from cancer or therapy, particularly cytotoxic drug therapy or radiotherapy), auto-immunity or otherwise. Septic shock is an illustration of a disease involving inflammation. Many of the clinical features of Gram-negative septic shock may be reproduced in animals by the administration of LPS to animals can prompt severe metabolic and physiological changes which can lead to death.
Associated with the injection of LPS is the extensive production of pro-inflammatory cytokines such as tuinour necrosis factor alpha (TNF.alpha.).
Cachexia, which is characteristic of chronic exposure to TNF or interleukin-6, is a common symptom of advanced malignancy and severe infection. It is characterised by abnormal protein and glucose metabolism and body wasting. Chronic administration of TNF IL-1 in mice, rats and/or humans cause anorexia, weight loss and depletion of body lipid and protein within 7 to 10 days (Cerami et al, 1985, Immunol. Lett, 11, 173: Fong et al, 1989 J. Exp. Med. 170, 1627. Moldawer et al, Am. J. Physiol., 254 G450-G456, 1988; Fong et am, Am. J Physiol. 256, R659-R665 (1989); McCarthy et al, Am. J. Clin. Nature. 42, 1179-1182. 1982). TNF levels have been measured in patients with cancer and chronic disease associated with cachexia.
TNF.alpha. and IL-1, with their common functional activities such as pyrogenicity, somnogenicity and being mediators of inflammation, have been implicated in the pathology of other diseases associated with chronic inflammation apart from toxic shock and cancer-related cachexia. TNF has been detected in synovial fluid in patients with both rheumatoid and reactive arthritis and in the serum of patients with rheumatoid arthritis (Saxne et al, 1988. Arthrit. Rheumat, 31, 1041). Raised levels of TNF have been detected in renal transplant patients during acute rejection episodes (Maury and Teppo 1987, J. Exp. Med. 166, 1132). In animals, TNF has been shown to be involved in the pathogenesis of graft-versus-host disease in skin and gut following allogenic marrow transplantation. Administration of a rabbit anti-murinie TNF antibody was shown to prevent the histological changes associated with graft-versus-host disease and to reduce mortality (Piquet et en, 1987, J. Exp. Med. 166, 1220). TNF has also been shown to contribute significantly to the pathology of malaria (Clark et al, 1987, Am. J. Pathol. 129, 192-199). Further, elevated serum levels of TNF have been reported in malaria patients (Scuderi et al, 1986, Lancet 2, 1364-1365).
PUFAs are known to have a range of useful biological activities (see for example International Patent Application Nos. WO 93/00084 and WO 95/00607 and the references cited therein). Unfortunately, due to their limited stability in vivo, PUFAs have not achieved widespread use as therapeutic agents. The present inventors have found that PUFAs including at least one .beta. oxa, .beta. thia, .gamma. oxa or .gamma. thia substitution have activity in a number of in vitro systems which suggest that these PUFAs may be useful in treatment of a range of disease states. In addition, the present inventors have developed substituted PUFAs which while retaining biological activity have increased stability in vivo ie slower metabolic turnover. The conjugation of an amino acid to PUFA also increases solubility of the compound.
The present inventors have also found that certain of the amino acid coupled PUFA suppress cytokine production and inhibit inflammation in response to carageenan and delayed type hypersensitivity to sheep red blood cells. Such compounds have utility in the treatment of T cell-mediated diseases, autoimmune disease, transplant rejection, graft vs host disease and allergic disease.
While saturated .beta. oxa fatty acids can be obtained using the standard procedure for ether synthesis, by reaction of alkyl halides with dianions of .alpha.-hydroxy acids or by treating .alpha.-halo acids with deprotonated alcohols, the unsaturated .beta.-oxa fatty acids of the present invention are not accessible using normal methods. Attempts to obtain the unsaturated compounds in this manner lead only to decomposition products, resulting from undesirable side reactions at the olefinic and allylic carbons.
In a recent variation of the standard procedure, saturated .beta.-oxa fatty acids have been obtained through nucleophilic substitution reactions under less vigorous conditions, by treating diazoacetates, activated by complexation with boron trifluoride etherate, with alcohols. However, boron trifluoride etherate is known to cause isomerization of alkenes and it is therefore unsuitable for use in the synthesis of unsaturated .beta.-oxa fatty acids.
As disclosed in co-pending International Patent Application No PCT/AU95/00677 (the disclosure of which is incorporated herein by cross reference) it has been found that unsaturated .beta.-oxa fatty acids can be obtained in good yields, by insertion of carbenes in the O--H bond of alcohols. There is no complication from other carbene insertion reactions and, of particular significance, the olefinic moieties are inert under the reaction conditions.
The carbene can be generated from the corresponding diazo acetate or diazo alkane, by treatment with a catalyst such as a rhodium salt. Reaction of the carbene with the complementary alcohol, which is either a derivative of .alpha.-hydroxy acetic acid or an unsaturated fatty alcohol affords the unsaturated .beta.-oxa fatty acid. In a preferred embodiment of the alcohols are those obtained by reduction of naturally occurring unsaturated fatty acids or the corresponding exters, and reaction with an ester of diazo acetic acid affords the unsaturated .beta.-oxa fatty acid.
It has also been shown that both .beta.-oxa and .beta.-thia substituted fatty acids are unable to undergo 1 oxidation. In addition, certain of these compounds display other properties which differ from those of natural PUFA including enhanced solubility, varied oxidation reduction potentials and different charge and polarity.